Project Summary Build-up of amyloid beta (A?) plaque in the brain is a hallmark feature of Alzheimer's disease. Research indicates that A? build-up is the result of inefficient clearance of A? as opposed to increased production. Breakdown by enzymes is one mechanism by which A? is cleared. Although several A? degrading enzymes have been identified, neprilysin (NEP) is the most physiologically relevant. Stimulating the activity of these enzymes is therefore a potential approach for the therapeutic manipulation of A? levels. However, at present there are no chemical compounds that can directly stimulate the activity of A? clearing enzymes. We have previously reported the discovery of a peptide (referred to as K49-P1-20) from a snake venom, which directly stimulates activity of NEP and its closest homolog endothelin converting enzyme (ECE). Our preliminary data indicate that K49-P1-20 can prevent the formation of A? plaque in a mouse model of Alzheimer's disease. Our published data indicate that replacing residue 9 of K49-P1-20 with Ala (Ala9K49-P1-20) can increase its potency and selectivity for NEP compared with ECE. Aim 1: To examine the selectivity of Ala9 K49-P1-20. We will examine the effect of increasing concentrations of Ala9 K49-P1-20 on the activity of each of the following enzymes which are also known to cleave A?: ECE-2, insulin degrading enzyme (IDE), angiotensin converting enzyme (ACE), MMP2 and MMP9. Initially enzyme activity will be measured using quenched fluorescent substrate based assays. Mass spectrometry will be used to determine the rate of A? cleavage by each of these enzymes in the presence or absence of Ala9 K49-P1-20. Aim 2: To determine the ability of Ala9K49-P1-20 to prevent the formation of A? plaques and associated behavioural impairments in a mouse model of AD. K49-P1-20 will be administered directly into the brain of B6C3-Tg(APPswe,PSEN1dE9)85Dbo mice via osmotic mini pumps. Drug infusion will begin prior to the formation of plaque and behavioral changes (at 4 months of age), and continue for 16 weeks. Mice will be subjected to the 8 arm radial maize test prior to treatment (T=0), and every 2 months thereafter in order to determine the effect of treatment on cognitive function. At the end of the treatment period, mice will be scarified, brain tissues removed and processed to determine amyloid beta levels. Above work will lay the foundation for a range of future studies which include understanding the mechanism behind enzyme stimulation, the development of peptidemimetics and most importantly the ability to REVERSE or halt the progression of Alzheimer's disease.